Cell-free DNA (cfDNA) in blood is used as a source of genetic material for noninvasive prenatal and cancer diagnostic assays in clinical practice. Recently we have started a project for new biomarker discovery with a view to developing new noninvasive diagnostic assays. While reviewing literature, it was found that exosomes may be a rich source of biomarkers, because exosomes play an important role in human health and disease. While characterizing exosomes found in human blood plasma, we observed the presence of cfDNA in plasma exosomes. Plasma was obtained from blood drawn into K3EDTA tubes. Exosomes were isolated from cell-free plasma using a commercially available kit. Sizing and enumeration of exosomes were done using electron microscopy and NanoSight particle counter. NanoSight and confocal microscopy was used to demonstrate the association between dsDNA and exosomes. DNA extracted from plasma and exosomes was measured by a fluorometric method and a droplet digital PCR (ddPCR) method. Size of extracellular vesicles isolated from plasma was heterogeneous and showed a mean value of 92.6 nm and a mode 39.7 nm. A large proportion of extracellular vesicles isolated from plasma were identified as exosomes using a fluorescence probe specific for exosomes and three protein markers, Hsp70, CD9 and CD63, that are commonly used to identify exosome fraction. Fluorescence dye that stain dsDNA showed the association between exosomes and dsDNA. Plasma cfDNA concentration analysis showed more than 93% of amplifiable cfDNA in plasma is located in plasma exosomes. Storage of a blood sample showed significant increases in exosome count and exosome DNA concentration. This study provide evidence that a large proportion of plasma cfDNA is localized in exosomes. Exosome release from cells is a metabolic energy dependent process, thus suggesting active release of cfDNA from cells as a source of cfDNA in plasma.

Using Streck's Cell-free DNA BCT tubes, it is possible to preserve the original proportion of fetal cell-free DNA for extended times as well as minimize the post-sampling maternal cell-free DNA background. Preserved in this way, fetal cell-free DNA can be amplified by WGA technology to be used in prenatal diagnostic tests.

Adenosine 3',5'-monophosphate, at a concentration of 40 micrograms per milliliter, inhibits the growth of HeLa and strain L cells in culture. The inhibition becomes progressively greater during the incubation of the cells. Adenosine 5'-monophosphate and adenosine, metabolites of adenosine 3',5'-monophosphate, do not affect the growth of either cell culture. This suggests that 3',5'-monophosphate enters the cell without alteration. Dibutyryl-adenosine 3',5'-monophosphate, reported to have a greater activity than adenosine 3'5'-monophosphate on several tissues, inhibited the growth of the cells much less.

After the injection of L-lysine and uniformly labeled L-lysine-carbon-14 into the rat, labeled homocitrulline and homoarginine are found in the liver and kidney. The ingestion of lysine by seven normal adults results in an increased urinary excretion of homocitrulline and homoarginine. These data suggest the occurrence of an unreported metabolic pathway for lysine in the rat and man.

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